This invention relates to flow microfluorometers and in particular to an improved flow microfluorometer wherein the entire fluorescence emission spectrum of particles can be rapidly obtained.
As is known, the fluoroscence spectrum of stained or unstained particles such as human cells can yield valuable information as to the nature of the cell and/or the condition of the cells. For example, the emission spectra of various stained particles within the blood are different. Thus, a count of the different types of white blood cells can be obtained since the emission spectra of these cells are different. Further, it is known the emission spectra of certain stained cancer cells are different from those of normal cells in certain tissues of the body. Again, the differences in emission spectra can be utilized to detect the abnormal presence of pathogenic cells.
It is known to accumulate spectra on individual cells by static single cell analysis or upon a stationary solution containing a number of cells, such as disclosed in U.S. Pat. Nos. 3,470,373; 3,497,690; 3,918,812; and 3,973,129. Further, it is known on a static basis to project a real image of a cell to a predetermined point, there being disposed at a point of rotating or oscillating wedge filter or grating whereby a continuous fluorescence emission spectrum of the static cell can be obtained. However, the accumulation of spectrum information is quite slow due to the static nature of the procedure. The use of a wedge filter in a spectrophotometer is also disclosed in U.S. Pat. No. 3,885,879.
Data can be more rapidly accumulated by dynamic single cell analysis in flow microfluorometers where cells are permitted to pass through an excitation light beam to effect fluoroscence thereof where, for example, 500-5,000 cells per second can flow through the beam. Such flow microfluorometers are disclosed, for example, in U.S. Pat. Nos. 3,586,859; 3,864,571; and 3,916,197. In some of the latter microfluorometers, at least two filters are employed, each of which passes a different wavelength of the fluorescence emissions spectrum of the activated particles. The filter outputs are then respectively focused at at least two different photomultiplier tubes. The outputs of the photomultiplier tubes are then combined for appropriate signal processing.
The resolution of the foregoing flow delivery systems is low in that only two wavelength portions or lines are detected out of the entire fluorescence emission spectrum of the particles. It is possible to increase this resolution by employing more filters and accordingly more photomultiplier tubes, there being, as indicated above, one photomultiplier tube for each filter. Of course, the resolution can be increased in this manner only to a certain point at which either space limitations or cost limitations occur. Not only is each photomultiplier tube expensive but also there are many applications where space is at a premium.
A further limitation of the above described microfluorometers is that the light supplied to the filters is divided between the filters. Hence, the light available at each filter output for detection is somewhat reduced thus rendering the system somewhat inefficient.